| Objective Helicobacter pylori(Hp) can invade gastric epithelial cells and multiply in the cells, then participate in the pathogenesis of Hp. But it was unclear about the mechanism of Hp invading gastric epithelial cells. Studies had confirmed that the integrin β1 mediated Hp invading cells, but it was unclear that which bacterial virulence factor associating with the invasion. In view of this, this study used Helicobacter pylori Cag A and Cag L which were known could bind to β1 integrin as a starting point to explore the influence of cag A and cag L to the invasive capability of Hp. The aim was to reveal the virulence factors associated with Hp invasion.Methods 1. Constructed Hp NCTC11637 cag L gene deletion strains. NCTC11637 cag L homology arms were designed by referencing Hp J99 genome sequence in NCBI. Using homologous recombination method, the kanamycin resistance gene(kanaR) was connected with gene fragments which were amplified by PCR from two end regions of cag L gene, and then co-inserted into p Bluescript SK Ⅱ(-). At last we constructed a mutational vector with kanamycin resistance marker. This mutational vector was transferred into NCTC11637 which contain complete cag L gene by electroporation. The cag L gene was replaced with kanamycin resistance gene by homologous recombination. We used kanamycin blood agar plate select NCTC11637 cag L gene deletion mutants, then named the mutants as NCTC11637Δcag L. The mutants were identified by Urease kit, PCR, and then cultured 25 generations for the verification of genetic stability. 2. The verification of β1 integrin expression in AGS and SGC7901Total RNA of two cell lines were extracted using Trizol, and then the c DNA was obtained by RT-PCR. We verified the m RNA expression levels of β1 integrin by PCR from the c DNA. We used membrane protein extraction kit to extract the cell total membrane protein, then verified the expression level of β1 integrin protein with anti-β1 integrin antibody by western blot. 3. The effects of cag A and cag L on Helicobacter pylori invading AGS and SGC7901. Using NCTC11637, NCTC11637Δcag L and NCTC11637Δcag A(constructed by our laboratory) infected AGS and SGC7901 for 2h at MOI(bacteria/ cell) = 50. Part of the cells were lysed and applied the lysate on blood agar plate for the count of bacterial which was the number of bacteria adherent cell. Another part of the cells were treated by gentamicin protection invasion assay for another 2h, then the cells were lysed and applied the lysate on blood agar plate for the count of bacterial which was the number of bacterial invasion. By calculating the invasion adhesion ratio:(the number of bacteria invaded cell / the number of bacteria adherent cell)×100%, we could know the effects of cag A and cag L on Helicobacter pylori invasion. 4. The affection of cag A and cag L on the time of Helicobacter pylori survival in cells Using NCTC11637, NCTC11637Δcag L and NCTC11637Δcag A infected AGS and SGC7901 for 2h(MOI = 50), then the cells were treated by gentamicin protection invasion assay and cultured with 25μg/ml gentamicin for 24 h, 36 h, 48 h, 72 h, 96 h. Then after the different time, we lysed the cells for bacterial counting.Results 1. Helicobacter pylori NCTC11637Δcag L was constructed successfully by homologous recombination gene knockout technology. After 25 generations, the NCTC11637Δcag L was proved stable by Urease kit and PCR. 2. AGS and SGC7901 could express β1 integrin, the expression of β1 integrin in AGS(6.387±2.957) was higher than SGC7901(4.442±2.382). 3. The results of the invasion adhesion ratio:(1) NCTC11637 invasion on AGS(28.285±4.646%) was stronger than SGC7901(0.194±0.038%);(2) In AGS NCTC11637, NCTC11637Δcag A, NCTC11637Δcag L invasion andadhesion ratio was 28.285±4.646%, 9.055±3.104% and 9.225±0.417%. NCTC11637 Δcag A, NCTC11637Δcag L invasion and adhesion were significantly lower than NCTC11637(P<0.05). There was no significant difference on invasion and adhesion between NCTC11637Δcag A and NCTC11637Δcag L;(3) In SGC7901 NCTC11637, NCTC11637Δcag A, NCTC11637Δcag L invasion and adhesion ratio was 0.194±0.038%,0.029±0.007% and 0.061±0.009%. NCTC11637 Δcag A invasion and adhesion ratio was significantly lower than NCTC11637(P<0.01), NCTC11637Δcag L invasion adhesion significantly lower than NCTC11637(P<0.05). 4.After infected with NCTC11637, NCTC11637Δcag A and NCTC11637Δcag L in AGS, the number of intracellular bacteria gradually reduced with the growth of the culture time. NCTC11637Δcag L could survive for 72 h, NCTC11637Δcag A and NCTC11637 could survive for 48 h.Conclusion 1. AGS and SGC7901 could express β1 integrin, β1 integrin in AGS was higher than SGC7901. 2. The invasion of Hp in AGS was stronger than SGC7901. 3. cag A and cag L was related with the invasion of Hp to cells. 4. After the deletion of cag A and cag L, NCTC11637 still could colonize and survive in cells. |
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